Francis J. Hughes

Polymorphisms in the IL-1A Gene are Correlated with Levels of Interleukin-1α Protein in Gingival Crevicular Fluid of Teeth with Severe Periodontal Disease

Interleukin-1 (IL-1) is a potent stimulator of bone resorption and is strongly implicated in the destruction due to bystander damage seen in periodontal disease. Recent studies suggest that polymorphisms of the (IL-1) gene complex may be significant risk factors for a number of chronic inflammatory diseases. The severity of periodontal disease has been positively associated with carriage of allele 2 at position -889 of the IL-1A gene in conjunction with allele 2 of the 1L-1B gene at position +3953. In this study, we tested the hypothesis that allele 2 of the IL-1A gene at position -889 might act to elevate levels of IL-la protein in patients with periodontal disease. Since levels of IL-la protein are low in healthy individuals, we used a group of patients with severe periodontal disease to investigate if levels of IL-1α protein in gingival crevicular fluid can be correlated to patient genotype. IL-la levels were measured by enzyme immunoassay in 46 patients with severe periodontal disease. These patients were genotyped by PCR and allele-specific restriction digests. The carriage rate for allele 2 in the diseased population was 68%. Overall, the carriage of allele 2 was associated with almost a four-fold increase in IL-la protein levels. Differences were most pronounced in non-smokers, while heavy smokers showed reduced levels of IL-1α protein regardless of genotype. These results suggest a mechanism whereby this genetic polymorphism acts to modulate IL-la protein production and may influence the pathogenesis of periodontal disease by affecting the extent of IL-1-associated bystander damage.

Recombinant Human Bone Morphogenetic Protein-2 Promotes Wound Healing in Rat Periodontal Fenestration Defects:

Although there is considerable interest in the use of bone morphogenetic protein (BMP) to promote periodontal regeneration, little is known of its effects on the early stages of wound healing. The aim of this study was to investigate the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an early stage of post-operative wound healing and following complete healing (10 and 38 days, respectively) in a rat model of periodontal regeneration. The buccal aspects of molar roots were carefully denuded of their periodontal ligament through a bony window created in the mandibles of Wistar rats under general anesthesia. After the root surfaces were acid-conditioned, a 10-μL quantity of 50 μg/mL rhBMP-2 in a collagen gel solution was placed into the surgically created defect in test animals; in controls, either a 10-μL quantity of only collagen gel was received, or the defect was untreated. Animals were killed 10 days or 38 days after surgery and the tissues processed for histological examination. Transverse 5-um sections were stained for the identification of new bone, cementum, and collagen fiber formation. In the 10-day study groups, new bone formation over the second molar and beyond the defect was significantly increased in the test group (p < 0.02), although there was no evidence of increased ankylosis. RhBMP-2 stimulated more than twice the area of cementum growth coronally compared with controls (712 ± 286 μm2 and 258 ± 57 μm2, respectively). Connective tissue attachment, including the number and width of collagen bundles, was similar in both test and controls. Complete healing without any evidence of ankylosis had occurred in all animals 38 days postoperatively, and no significant differences were observed between test and control groups. In conclusion, a single dose of rhBMP-2 increased the rate of normal intramembranous bone formation and selectively enhanced cementum formation coronally during early wound healing. However, the finding that rhBMP-2 induced bone formation at some distance from the defect suggests the importance of developing a suitable delivery system to maintain the concentration of BMP-2 at the site of implantation for potential therapeutic use.

Effects of Medium Supplements on Proliferation, Differentiation Potential, and In Vitro Expansion of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) possess great potential for use in regenerative medicine. However, their clinical application may be limited by the ability to expand their cell numbers in vitro while maintaining their differential potentials and stem cell properties. Thus the aim of this study was to test the effect of a range of medium supplements on MSC self‐renewal and differentiation potential. Cells were cultured until confluent and subcultured continuously until reaching senescence. Medium supplementation with fibroblast growth factor (FGF)‐2, platelet‐derived growth factor (PDGF)‐BB, ascorbic acid (AA), and epidermal growth factor (EGF) both increased proliferation rate and markedly increased number of cell doublings before reaching senescence, with a greater than 1,000‐fold increase in total cell numbers for AA, FGF‐2, and PDGF‐BB compared with control cultures. Long‐term culture was associated with loss of osteogenic/adipocytic differentiation potential, particularly with FGF‐2 supplementation but also with AA, EGF, and PDGF‐BB. In addition FGF‐2 resulted in reduction in expression of CD146 and alkaline phosphatase, but this was partially reversible on removal of the supplement. Cells expressed surface markers including CD146, CD105, CD44, CD90, and CD71 by flow cytometry throughout, and expression of these putative stem cell markers persisted even after loss of differentiation potentials. Overall, medium supplementation with FGF‐2, AA, EGF, and PDGF‐BB greatly enhanced the total in vitro expansion capacity of MSC cultures, although differentiation potentials were lost prior to reaching senescence. Loss of differentiation potential was not reflected by changes in stem cell surface marker expression.

Oral malodour--a review.

Halitosis is a very common condition which may affect up to 30% of the population. In most cases the aetiology of the condition is from local oral causes (oral malodour). Oral malodour is the result of the action of anaerobic bacteria in producing a range of malodorous molecular species including volatile sulphur compounds. Whilst malodour is often associated with the presence of periodontitis, in many cases there is no such link, and the evidence points to the importance of these anaerobic bacteria in tongue coatings which results in the clinical presentation of oral malodour. Management of oral malodour is directed at managing and reducing the bacterial load both in periodontitis and in tongue coatings by instituting proper oral hygiene measures, control of tongue flora by brushing or scraping, and possibly the adjunctive use of antiseptic agents. Treatments have also been proposed to neutralise malodorous compounds by chemical agents to mask the presence of the condition. Further evidence is required to demonstrate the long-term efficacy of therapies for this troublesome condition.

Inhibition of Akt/mTOR Attenuates Age‐Related Changes in Mesenchymal Stem Cells

The decline in mesenchymal stem cell (MSC) self‐renewal and function with aging contributes to diseases associated with impaired osteogenesis. MSC donor age in prolonged culture also limits the therapeutic potential of these cells for tissue engineering and regenerative medicine. Here, we demonstrate an intervention to preserve the immature state MSC and consequently maintain self‐renewal and differentiation capacity during in vitro aging. We showed that blocking of phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (mTOR) prevents the development of an age‐related phenotype and maintains MSC morphology of early passage cells with high clonogenic frequency and enhanced proliferative capacity. MSC cultured in the presence of inhibitors of Akt or mTOR also robustly maintain their osteogenic potential, that is otherwise lost during in vitro aging. We further report that these effects may be mediated by induction of expression of pluripotency genes Nanog and Oct‐4 and by the reduction in the production of cytoplasmic reactive oxygen species (ROS). Additionally, loss of Akt/mTOR and ROS was accompanied with lower levels of DNA damage. These results provide an insight into mechanisms involved in MSC aging and suggest possible interventions to maintain quiescence and function of MSC prior to in vivo transplantation or as pharmacological agents in diseases associated with loss of MSC function. Stem Cells 2014;32:2256–2266

Akt- and Erk-mediated regulation of proliferation and differentiation during PDGFRβ-induced MSC self-renewal.

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.

Diabetes and periodontal disease: a two-way relationship

Periodontitis and diabetes are common, complex, chronic diseases with an established bidirectional relationship. That is, diabetes (particularly if glycaemic control is poor) is associated with an increased prevalence and severity of periodontitis, and, severe periodontitis is associated with compromised glycaemic control. Periodontal treatment (conventional non-surgical periodontal therapy) has been associated with improvements in glycaemic control in diabetic patients, with reductions in HbA1c of approximately 0.4% following periodontal therapy. For these reasons, management of periodontitis in people with diabetes is particularly important. The dental team therefore has an important role to play in the management of people with diabetes. An emerging role for dental professionals is envisaged, in which diabetes screening tools could be used to identify patients at high risk of diabetes, to enable them to seek further investigation and assessment from medical healthcare providers.

Involvement of Autoimmunity in the Pathogenesis of Aggressive Periodontitis

The aim of this study was to investigate the involvement of autoimmune reactions to native and post-translationally modified extracellular matrix components in the pathogenesis of periodontitis. Sera from individuals with aggressive periodontitis (AgP, n = 25), chronic periodontitis (CP, n = 14), and gingivitis (G, n = 18) were tested for the presence of autoantibodies against: (a) native collagen type I (CI) and collagen type III (CIII); (b) CI and CIII post-translationally modified by reactive oxygen species (ROS) of the type present during inflammation; and (c) citrullinated filaggrin-derived peptides (CCP). Autoantibodies to native and ROS-modified CI and CIII as well as autoantibodies to CCP were observed exclusively in patients with AgP and not in those with CP or G. In conclusion, autoimmune reactions to native and post-translationally modified self-antigens may play a role specifically in the pathogenesis of AgP.

Adult rat bones maintain distinct regionalized expression of markers associated with their development.

The incidence of limb bone fracture and subsequent morbidity and mortality due to excessive bone loss is increasing in the progressively ageing populations of both men and women. In contrast to bone loss in the weight-bearing limb, bone mass in the protective skull vault is maintained. One explanation for this could be anatomically diverse bone matrix characteristics generated by heterogeneous osteoblast populations. We have tested the hypothesis that adult bones demonstrate site-specific characteristics, and report differences at the organ, cell and transcriptome levels. Limb bones contain greater amounts of polysulphated glycosaminoglycan stained with Alcian Blue and have significantly higher osteocyte densities than skull bone. Site-specific patterns persist in cultured adult bone-derived cells both phenotypically (proliferation rate, response to estrogen and cell volumes), and at the level of specific gene expression (collagen triple helix repeat containing 1, reelin and ras-like and estrogen-regulated growth inhibitor). Based on genome-wide mRNA expression and cluster analysis, we demonstrate that bones and cultured adult bone-derived cells segregate according to site of derivation. We also find the differential expression of genes associated with embryological development (Skull: Zic, Dlx, Irx, Twist1 and Cart1; Limb: Hox, Shox2, and Tbx genes) in both adult bones and isolated adult bone-derived cells. Together, these site-specific differences support the view that, analogous to different muscle types (cardiac, smooth and skeletal), skull and limb bones represent separate classes of bone. We assign these differences, not to mode of primary ossification, but to the embryological cell lineage; the basis and implications of this division are discussed.

The presence, function and regulation of IL-17 and Th17 cells in periodontitis.

Periodontitis (PD) is a chronic inflammatory disease characterised by tissue inflammation and destruction of the associated alveolar bone. It is caused by the colonisation of the bacterial plaque biofilm and the resultant host immune responses in the surrounding periodontal tissues. The pro-inflammatory cytokine IL-17, and IL-17 producing CD4+ T cells (also called Th17 cells) have been shown to play an important role in many inflammatory diseases. There is increasing evidence of the presence of IL-17 and Th17 cells in human PD lesions and this may be associated with disease severity. Moreover, several animal studies indicate the potential role of IL-17 and Th17 cells in gingival inflammation and the resultant bone destruction in PD. Here we review recent findings regarding the presence, function and regulation of IL-17 and Th17 cells in PD, and we highlight potential areas of future research interest.